WO2001096531A1

WO2001096531A1 - Novel streptomyces sp. wl-2 strain producing xylanase

Info

Publication number: WO2001096531A1
Application number: PCT/KR2001/001014
Authority: WO
Inventors: Ki-Haeng Cho; Ki-Hong Yoon; Dae-Weon Kim; Hwa-Gyun Oh; Eun-Hee Lee
Original assignee: Ctc Bio, Inc.
Priority date: 2000-06-14
Filing date: 2001-06-13
Publication date: 2001-12-20
Also published as: KR20010111986A; AU2001266366A1; KR100411771B1

Abstract

The present invention relates to a Streptomyces sp. WL-2 strain producing a xylanase and the neutral xylanase produced by the strain. The present invention comprises of isolating the Streptomyces sp. strain from the soil, identifying the strain, incubating said strain in culture media containing α-cellulose and maltose or oat xylane and maltose, and producing the xylanase in large quantities.

Description

NOVEL STREPTOMYCES SP. WL-2 STRAIN PRODUCING XYLANASE

TECHNICAL FIELD

The present invention relates to a novel Streptomyces sp. WL-2 strain which produces xylanase. More specifically, this invention relates .to a production of xylanase for a feed additive for monogastric animals by isolating novel Streptomyces sp. strains from the soils, identifying and culturing the said microorganism wherein the xylanase has high activity in neutral medium.

BACKGROUND ART

A Xylanase is amongst various enzymes which are now being produced from a microbe and have an expectation to develop useful applications in the future. The major feature of the enzyme is its ability to degrade plant cellulose materials, (the biomass that is most common on earth and renewable by solar energy), convert them into sugar, and turn into energy resources. Practical use will be made possible if more research is completed. At present, the applications of xylanase include the production of high-grade paper, the regeneration of waste paper, impurity removal from fruit beverages and beer, extract of coffee, plant oil and starch, and industrial applications such as improvement of feed utilization for monogastric animals, which is largely the focus of the present invention.

Recently, the usefulness of xylanase as an enzyme for feed additives has been increasing. Feed grains, which provides a saccharide and a small amounts of protein needed for the energy metabolism of animals, contain fiber that most monogastric animals cannot use. The dietary fiber of grains is composed of non- starch polysaccharides and lignins. The cell wall components of plants are made of by joining lignins and non-starch polysaccharides composed of layers of cellulose on the outside of the cell, hemicellulose protecting its internal whole-grain and beta- glucans. The non-starch polysaccharides decrease efficiency that livestock uses a nutrient due to protecting starch and protein in grain as well as being difficult to digest by livestock digestive enzymes. Furthermore, the non-starch polysaccharides is dissolved in a sticky state by the viscous gel in the small intestine, and that digestive enzyme hinders the approach of nutrients and obstructs the flow of digestion material in the alimentary canal. Therefore, a microbe attached to the digestion materials creates abnormal fermentation and results in loss of nutrient.

If xylanase is added to grain feed, the hemicellulose layers protecting the grains are hydrolyzed and the availability of nutrient from the grain is increased. In addition, the condition of digestion material in livestock intestine can be greatly improved.

Particularly, in the example of using wheat having high hemicellulose content as feed, the phenomenon of Serpulina hyodysenteriae or dysentery in livestock occurs. However the xylanase prevents the above phenomenon, and enhances the value as a feed. Therefore, diversification of feed resources and improvement of feeding efficiency for the breeding of livestock are very important issues, so the xylanase is a very important enzyme and feed additive for monogastric animals. Animal consumption of animals such as pigs, poultry have recently increased countries where living standards are also increasing.

The microbe used in production of xylanase for a feed additive is an example of a fungus. Trichoderma sp. strain of fungus is mainly used, of which enzyme productivity is generally excellent compared with the enzyme productivity of bacteria producing xylanase and the strain shows maximum activity in the acid condition. The physiological condition of an alimentary canal of pigs or poultry is different and distinct on each parts, but the pH condition next to a small intestine, on which xylanase has to act, is near 6.5. Therefore, xylanase, which has a high activity in a nearly neutral condition, is required more as an enzyme for a feed additive, than that which be derived from a fungus having high activity with a pH of around 5.0.

There are a various kinds of Bacteria producing xylanase having high activity in a nearly neutral condition wherein the bacteria are belonging to Aeromonas sp., Bacillus sp., Clostridium sp., Streptomyces sp. and so on, and reactive characteristics of xylanase produced from the bacteria are various. Also, three kinds of enzymes, endo-xylanase, exo-xylanase and xylosidase, are known to act together to completely degrade xylan(major components of hemicellulose), convert them sugar and they are commonly called xylanase-type enzymes. On the other hand, the present invention places emphasis on the first endo- xylanase of the enzymes and from now on, the enzyme is called xylanase for the sake of convenience. However, it has never been reported about the method of how to produce xylanase that has high activity in neutral acidity. Accordingly, the present inventors have now completed the invention through isolating novel Streptomyces sp. strains from the soil, producing xylanase having high activity in neutral acidity, identifying them, finding that alpha-cellulose, xylan, maltose and etc. enhance enzyme productivity of the isolated strain, culturing the above strain and producing in large quantities the xylanase valuable for a feed additive.

Therefore, the objective of this invention is to provide novel Streptomyces sp. strains which producing xylanase, isolated and identified from the soil. The other objective of this invention is to provide a method of mass production of xylanase which has a high activity in neutral acidity, which is available as a feed additive by culturing the above Streptomyces sp. strain. A further objective of this invention is to provide xylanase added livestock feed produced by the above method. The above objective of this invention has been achieved through isolating numerous actinomicetes from the soil, inoculating them in a liquid medium, collecting strain having high xylanase activity, invesgating mycological characteristics of the collected strain and characteristics of the produced xylanase. This also includes dialyzing productivity of xylanase and decomposition power of wheat bran, wheat by-product through xylanase, depending on culture medium composition of the collected strain, thereby confirming availability as for a feed additive and producing the feed.

DISCLOSURE OF INVENTION

This invention comprises of inoculating each antinomicetes colony, which is formed by putting soil samples in medium for antinomicetes isolation, in the composite medium to cultivate them at 28 °C for 4 days. Then by using a cultivation- equivalent solution as an crude enzyme solution, and choosing strains excellent at decomposing oat xylan and selecting microbes producing xylanase. The next step is to examine and identify the morphological, cultural, and biochemical properties of the above selected strains whilst letting the xylanase, which is an enzyme produced by the Streptomyces sp. WL-2 strain, conduct enzymatic reactions with different reaction temperatures and pHs to investigate optimum activity temperature and pH.

This then determines the optimum temperature and pH for activation in regards to cultivating the Streptomyces sp. WL-2 strain at the medium to which oat xylan and alpha-amylose, a high molecular carbon source, are added. Then comes the examination of the activation of xylanase and determining the influence of the carbon source on productivity of xylanase.

By adding a different kind of carbon source instead of glucose in the medium in the above step, we cultivate the Streptomyces sp. WL-2 strain and examine the activation of xylanase at regular intervals and determine the productivity of xylanase. After processing the xylanase enzymes produced by the Streptomyces sp. WL-2 strain with bran, a by-product of wheat, we examine the increment of reducing sugar produced through xylanase activation, and this determines the usefulness of xylanase produced by the Streptomyces sp. WL-2 strain as a feed additive, and for producing livestock feed.

In the above step, The medium for isolating antinomicetes from the soil sample is composed of humic acid lg L, Na₂HPO 0.5g/L, KC1 1.7g/L, MgSO₄- 7H₂O 0.05g/L, FeSO₄ ^• 7H₂O O.Olg/L, CaCO₃ 0.02g/L, Thiamine 0.5mg/L, Riboflavin 0.5mg/L, Niacin 0.5mg/L, Pyridoxine 0.5mg/L, Inositol 0.5mg/L, Calcium pan tonic acid 0.5mg/L, Para-amino benzoic acid 0.5mg/L, Biotin 0.25mg/L, Cyclohexamid 50mg/L, Nalidicsic acid 50mg/L, Agar 18g/L, and humic acid-vitamin(HV) agar medium controlled with pH 7.2 was used. The medium for culturing antinomicetes is composed of soluble starch lOg/L, Glucose 20g/L, Soybean oil cake 25g/L, meat extract lg/L, Yeast extract 4g/L, NaCl 2g L, K₂HPO 0.25g/L, CaCO₃ 2g/L, and G.S.S composite medium controlled with pH 7.2 was used. The identification of Streptomyces sp. Strain was carried out in accordance with Bergey's mamual of determinative bacteriology and ISP (International Streptomyces Project).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electron microscope photograph of Streptomyces sp. WL-2 strain according to the present invention.

FIG. 2 is a graph showing activity depending on reaction temperature and pH of neutral xylanase produced from Streptomyces sp. WL-2 strain according to the present invention

FIG. 3 is a graph showing neutral xylanase activity depending on culturing time, respectively culturing in prepared medium that Streptomyces sp. WL-2 strain according to the present invention varies its culture medium and carbon sources

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in detail in connection with preferred embodiments. The below embodiments are provided for the sake of clear understanding only and the present invention is not limited thereto.

Example 1 : Isolating and selecting strains producing xylanase from the soil.

To isolate the antinomicetes producing xylanase according to this invention, we suspended 1 g of soil sample into 10 ml of the physiological saline solution, and then placed an adequate amount of the suspended solution on the humic acid- vitamin(HV) agar to isolate antinomicetes to cultivate at 28 °C for 7 days. Among the colonies formed at the solid agar, those with different forms were inoculated in the G.S.S. composite liquid medium for antinomicetes cultivation to cultivate at 28 ^°C for 4 days.

Using a cultivation-equivalent solution acquired through centrifugal separation of the antinomicetes culture solution, we examined the xylanase activations to finally select the strain most useful and relevant in xylanase activation.

Example 2: Identifying the Streptomyces sp. WL-2 strain and examining its characteristics

We examined and identified the morphological, cultural, and physiological characteristics of the strains isolated and selected in the above example 1. By cultivating the above strains at various temperatures, we determined the temperature range possible for cultivation.

As a result the morphological, cultural, and physiological characteristics of the selected strains are shown in table 1, 2, and 3. The spore chain and spore shape of the strains after cultivation on the solid medium for 21 days is shown in Fig 1. In particular, judging from its ability to produce LL-diaminopimelic acid, this invention of the actinomicetes strain, can be identified through Streptomyces sp. WL-2 strain among actinomicetes. In addition, the identified Streptomyces sp. WL-2 strain did not grow at temperatures over 35 °C, and yet showed normal growth at temperatures between 10 and 30°C, which shows it grows well at the lower temperature. Therefore, this invented strain as selected above was named the Streptomyces sp. WL-2 strain and deposited at the gene bank in the Korea Research Institute Of Bioscience and Biotechnology with the No. KCTC 0801BP on June 7, 2000.

Table 1 : Morphological characteristics oϊ Streptomyces sp. WL-2 strain of the present invention

Table 2: Cultural characteristics of Streptomyces sp. WL-2 strain from the present invention

Table 3: Physiological characteristics of Streptomyces sp. WL-2 strain from the present invention

Example 3; Characteristics examination of xylanase produced from

Streptomyces sp. WL-2 strain of the present invention

To investigate activity depending on reaction temperature and pH in a hydrolysis of oat xylan by using xylanase produced from Streptomyces sp. WL-2 strain of the present invention, a cultivation-equivalent solution was concentrated and fractioned with ammonium sulfate to produce a precipitate, suspended in 20 mM Na- phosphate buffer solution(pH 7.0), and then dialyzed with the same buffer solution. The fraction having xylanase activity of the fractioned protein was collected to use as a crude enzyme solution and the xylanase activity was measured, varying reaction temperature and pH As shown in Fig. 2, maximum activity was showed in a nearly 60 °C and pH

6.0 and activity more than 90% in the range of pH 4.5 to 6.5, especially more than 95% in pH 6.5. Therefore, it is found that xylanase produced from Streptomyces sp. WL-2 strain of the present invention has excellent activity in a nearly neutral condition, which is suitable for strain cultivation of the present invention. Also, we confirmed that xylanase activity of enzyme sample is base on xylanase activity, as enzyme sample used in reaction did not show xylosidase activity. Example 4: Xylanase production using Streptomyces sp. WL-2 strain of the present invention by a high molecule saccharide addition

By respectively adding various kinds of high molecule saccharide to medium, xylanase productivity using Streptomyces sp. WL-2 strain of the present invention was determined. As a result, when alpha-cellulose and oat xylan was added to the medium, we confirmed that xylanase productivity increases, and then examined the influence of addition quantity of alpha-cellulose and oat xylan in medium on productivity of xylanase of Streptomyces sp. WL-2 strain. G.S.S medium was employed as control sample, and Streptomyces sp. WL-2 strain of the present invention was inoculated into liquid medium adding alpha-cellulose or oat xylan of a various concentration instead of soluble starch in the medium, cultured at 28 ^°C for 4 days, and then xylanase activity of cultivation-equivalent solution was examined

As shown in Fig. 4, medium of addition of 1.0% for alpha-cellulose showed the highest enzyme productivity and the increment of enzyme production was 8 times as large than control sample. In certain case of addition of oat xylan 1.5%, the enzyme was produced in 9 times larger amount.

Table 4: Influence of addition quality of alpha-cellulose or oat xylan on enzyme productivity

Example 5: Xylanase production using Streptomyces sp. WL-2 strain of the present invention by sugar addition

Using the results obtained from the above example 4, the medium adding alpha-cellulose(l%) or oat xylan(1.5%) in place of soluble starch eliminated in G.S.S medium was employed as control sample, and Streptomyces sp. WL-2 strain of the present invention was inoculated into medium(adding 1% of maltose, arabinose and galactose respectively in place of glucose of G.S.S medium component) to cultivate them at 28 °C for 4 days, xylanase productivity was examined, and then maltose was added in the medium, thereby confirming maximum xylanase productivity. Therefore, the strain of the present invention was inoculated into medium, varying addition quality of maltose, cultured them, and xylanase activity was measured on a cultivation-equivalent solution.

As shown in Fig.5, medium adding 1% of maltose has maximum enzyme productivity, and xylanase activity was increased more than that of control sample containing glucose. Especially, in medium containing alpha-cellulose, the increment of enzyme production by maltose(l%) addition was higher than that containing oat xylan, and 14 times increased compared with control sample.

Table 5: Influence of addition quality of maltose on enzyme productivity

Example 6: Xylanase production of Streptomyces sp. WL-2 strain of the present invention depending on culturing time

Streptomyces sp. WL-2 strain of the present was respectively inoculated into

G.S.S medium which is typical medium of Streptomyces sp. strain and G.S.S alpha medium and G.S.S xylan medium respectively containing maltose 1% obtained from the example 5, and xylanase activity was examined, shake- culturing them at 28 ^°C for 5 days.

As shown in Fig.3, xylanase activity of a culture solution was increased until culturing time has comes to 4.5 days, after that time, slight decrease was shown and maximum xylanase activity in G.S.S alpha medium adding maltose 1%.

Therefore, the strain of the present invention was cultivated for 4.5 days in G.S.S alpha medium to obtain the xylanase of about 120 unit/mL.

Example 7: Examination of usefulness for feed addition of xylanase obtained from Streptomyces sp. WL-2 strain of the present invention

To examine whether xylanase obtained from Streptomyces sp. WL-2 strain of the present invention was adequate for a feed additive, xylanase derived from strain by the present invention was treated with bran of wheat by-product, and then the quantity of reducing sugar obtained by enzyme activity was analyzed. Above all, treptomyces sp. WL-2 strain was cultivated in G.S.S alpha medium, and a cultivation-equivalent solution was precipitated with ammonium sulfate to obtain crude enzyme solution, and feed for a livestock was produced by adding to 1 gram of bran to get xylanase 100 unit/mL, followed by reacting them in pH 6.5 and 37°C, the physiological condition of small intestine of monogastric animals. As shown in Fig. 6, the bran was decomposed depending on reaction time and the value of reducing sugar in reaction solution was increased. Considering these results, we found out that xylanase derived from strain of the present invention is suitable for feed additive. Table 6: Bran decomposition power of xylanase obtained from Streptomyces sp. WL-

2 strain of the present invention

INDUSTRIAL APPLICABILITY

As explained through the above embodiments, Streptomyces sp. strain is isolated and identified from the soil, named Streptomyces sp. WL-2 strain. The neutral xylanase, which is obtained by culturing in medium containing alpha- cellulose and maltose or oat xylane and maltose, is suitable for feed additive and useful as a substitute of acidic xylanase. The xylanase of the present invention is very useful in the feed, fertilizer and paper-making industries because remarkable effects show in the field of paper manufacturer, regeneration of waste paper and manure fermentation.

While the present invention has been described with reference to the particular illustrative embodiments, it is not to be restricted by the embodiments but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiments without departing from the scope and spirit of the present invention. Applicant's or agent's file International application No. reference number YL01012PCT PCT/KR01/01014

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bis)

A. The indications made below relate to the microorganism referred to in the description on page 7 , line 16-19 .

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet I I

Name of depositary institution

Korean Collection for Type Cultures (KCTC)

Address of depositary institution (including postal code aid country)

Korea Research Institute of Bioscience and Biotechnology (KRIBB) #52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea

Date of deposit Accession Number

2000. 6. 12. KCTC 0801BP

C. ADDITIONAL INDICATIONS (leave blank if not applicable)

This information is continued on an additional sheet □

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated states)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e.g., "Accession Number of Deposit")

Claims

WHAT IS CLAIMED IS:

1. A Streptomyces sp. WL-2 strain (KCTC 0801BP) producing neutral xylanase.

2. A method for producing a neutral xylanase, comprising the steps; inoculating the strain of said claim 1 into G.S.S composite liquid medium for antinomicetes cultivation, the improvement which was added with 0.1 — 10% of alpha-cellulose or oat xylan respectively in place of soluble starch and with 0.1 ~ 10%) of maltose in place of glucose, and culturing said strain at 28 °C for 4 — 5 days.

3. A neutral xylanase derived from Streptomyces sp. WL-2 strain (KCTC 080 IBP) having decomposition power of hemicellulose, produced by said claim 2 method and showed enzyme activity in the range of 50 — 70 °C temperature and pH 4-8.

4. A feed for monogastric livestock adding the neutral xylanase of said claim 3.